MESOSCALE NANOPARTICLES FOR SELECTIVE TARGETING TO THE KIDNEY AND METHODS OF THEIR THERAPEUTIC USE
A drug carrier nanoparticle has been synthesized that can specifically target the proximal tubules of the kidneys. The nanoparticles accumulate in the kidneys to a greater extent than other organs (e.g., up to 3 or more times greater in the kidney than any other organ). They can encapsulate many classes of drug molecules. The nanoparticles are biodegradable and release the drug as they degrade. The particles can sustainably release a drug within the kidneys for up to two months. The nanoparticles are useful for the treatment of diseases that affect the proximal tubules, such as heart failure, liver cirrhosis, hypertension, and renal failure; the study of relative blood flow to the renal cortex and medulla; and delivery of agents to treat gout.
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This application is a continuation of U.S. patent application Ser. No. 15/558,970, filed Sep. 15, 2017, which is a National Stage Application of PCT/US2016/022879, filed Mar. 17, 2016, which claims the benefit of and priority to U.S. Provisional Patent Application No. 62/136,104, filed Mar. 20, 2015, the entire contents of which are incorporated herein by reference.
GOVERNMENT FUNDINGThis invention was made with government support under Grant No. DP2-HD075698 awarded by National Institutes of Health. The government has certain rights in this invention.
FIELD OF THE INVENTIONThis invention relates generally to nanoparticles and methods of their manufacture and therapeutic use. In particular embodiments, the invention relates to mesoscale nanoparticles for selective targeting of the renal proximal tubule epithelium.
BACKGROUNDSpecific physiological parameters that enhance delivery to diseases sites, including the enhanced permeability and retention (EPR) effect to localize nanoparticles in tumors, have been exploited to target specific sites in tissues. However, the EPR effect has not yet been shown to result in significant targeting in human patients, possibly due to low accumulation in small tumors and disseminated disease. Recently, “active” targeting of disease sites have been investigated via functionalization of nanoparticles with molecular recognition moieties such as antibodies, small molecules, or aptamers. This general approach has generated positive pre-clinical results, some of which have progressed to clinical trials.
Often, and sometimes irrespective of a molecular targeting element, nanoparticles may localize in one of several organs due to the particle surface chemistry, size, and zeta potential. The purposeful application of this mechanism may allow for the treatment of diseases regardless of the expression of molecular targets or the size of a lesion. The delivery of targeted agents to specific organs and tissues may obviate off-target effects in systemic delivery such as neutropenia or GI toxicity. To employ this targeting approach, there is a need to understand the properties of nanoparticles that cause differential biodistribution in specific organs and cell types.
Several kidney diseases may benefit from the development of nanoparticle therapeutics which allow for site-directed accumulation, controlled temporal release, and protection of a therapeutic payload. Among candidate diseases are lupus, glomerulonephritis, and renal cell carcinoma (RCC), which often arises in the proximal tubules. Pharmacological therapeutic options for these diseases are limited, thus there is a strong need to increase the efficacy and decrease side effects of current drugs.
Only a few instances of previous work have attempted to specifically target the kidney using nanoparticles. First, one technology was limited to kidney targeting where accumulation in the kidney is only marginally greater than the next highest organ and experiences rapid degradation in vivo (Fischer et al. PLOS ONE 2014 9: e93342 and Shenoy et al. Pharmaceutical Research 2005 22: 2107-2114). A second technology synthesized nanoparticles that can accumulate in the glomerulus of kidneys (Choi et al. PNAS 2011 108: 6656-6661). However, these nanoparticles exhibit significantly greater accumulation in the liver and spleen compared to the kidney. While other examples of nanoparticle technologies synthesized with PLGA-PEG exist, none accumulates in the kidneys. Moreover, although low-molecular weight polymers, peptides, and proteins have been shown accumulate in kidney proximal tubules, these platforms are quickly cleared from the body and do not carry a large therapeutic payload (Dolman et al Advanced Drug Delivery Reviews 2010 62:1344-1357).
Therefore, there is a strong need for nanoparticles that specifically target and accumulate in the kidney, exhibit high stability over a long period of time, and provide the ability to carry and deliver large therapeutic payloads to the kidney.
SUMMARY OF INVENTIONDescribed herein are mesoscale nanoparticles for selective targeting to the kidney. A drug carrier nanoparticle has been synthesized that can specifically target the proximal tubules of the kidneys. The nanoparticles accumulate in the kidneys to a greater extent than other organs (e.g., up to 3 or more times greater in the kidney than any other organ). They can encapsulate many classes of drug molecules. The nanoparticles are biodegradable and release the drug as they degrade. The particles can sustainably release a drug within the kidneys for up to two months. The nanoparticles are useful for the treatment of diseases that affect the proximal tubules, such as heart failure, liver cirrhosis, hypertension, and renal failure; the study of relative blood flow to the renal cortex and medulla; and delivery of agents to treat gout.
Because of the kidney-targeting ability of the drug carrier nanoparticles, treatment of the kidney can now be performed using therapeutic agents that are currently not used for kidney treatment and/or that are currently experimental. Various therapeutic agents (e.g., TGFb inhibitors, mTOR inhibitors, everolimus, kinase inhibitors) have not been usable for kidney disease treatment due to poor pharmacokinetics. By the time the kidneys respond to a delivered therapeutic agent, toxicity side-effects occur elsewhere in the body. Accordingly, the mesoscale nanoparticles disclosed herein can selectively deliver therapeutic agents (e.g., therapeutic agents that are typically not used for kidney disease treatment due to poor pharmacokinetics) to the kidney while minimizing toxicity elsewhere in the body.
In one aspect, the invention is directed to a mesoscale nanoparticle composition comprising: a core (e.g., polymer, poly(lactic-co-glycolic acid) (PLGA), silica, chitosan, lipid, liposome, metal e.g., gold, iron oxide); and a surface coating (e.g., polyethylene glycol, non-opsonizing lipid) having a surface charge between −40 mV to +40 mV (or −25 mV to +25 mV, or −15 mV to +15 mV), wherein the composition is in the form of a nanoparticle having a diameter (e.g.; average diameter) from 200 nm to 1000 nm (or 250 nm to 500 nm) (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution).
In certain embodiments, the core comprises PLGA. In certain embodiments, a base of PLGA is modified. In certain embodiments, the PLGA has a molecular weight from 7 kDa to 54 kDa. In certain embodiments, the surface coating (e.g., polyethylene glycol, non-opsonizing lipid) has a molecular weight from 2 kDa to 10 kDa (e.g., from 4 kDa to 7 kDa, e.g., 5 kDa). In certain embodiments, the surface coating is selected from the group consisting of polyethylene glycol (PEG), PEG-carboxylic acid, PEG-carboxylic acid-DMAB, and methoxy PEG. In certain embodiments, the weight ratio of PEG to PLGA is from 9% to 13% (e.g., prior to synthesis of the mesoscale nanoparticle composition).
In certain embodiments, the mesoscale nanoparticle composition comprises a therapeutic agent (e.g., a hydrophobic small molecule; a targeted chemotherapeutic (e.g., doxorubicin, sorafenib, or sunitinib); a metabolic targeting therapeutic (e.g., STF-31, CPI-613, or Fasentin); alpha-7 nicotine receptor antagonist; a hypertension therapeutic; a TGFbeta inhibitor (e.g., kinase inhibitors (e.g., LY2157299 (galunisertib), SD-208, SB505124)); a reactive oxygen species and DNA damage inhibitor (e.g., Amifostine (Ethyol)), a Nf kappa B inhibitor (e.g., Pyrrolidine dithiocarbamate, quinazoline, BMS-345541, BAY-11-7085); a p21 inhibitor (e.g., siRNA or small-molecule inhibitor); a mTOR inhibitor (e.g., RAD001 (everolimus)); a glutaminase inhibitor (e.g., BPTES, CB-839); a therapeutic as listed in Table 3; and/or a diuretic).
In certain embodiments, the therapeutic agent is noncovalently associated with the nanoparticle, e.g., encapsulated within the surface coating. In certain embodiments, the therapeutic agent is attached to the nanoparticle (e.g., non-covalently attached or covalently bound to a moiety of the surface coating).
In certain embodiments, the mesoscale nanoparticle composition comprises an imaging agent (e.g., a dye, e.g., a fluorescent molecule, e.g., a fluorescent molecule within the core). In certain embodiments, imaging agent is selected from the group consisting of a near infrared dye (e.g., 3,3′-Diethylthiadicarbocyanine iodide) and a far-red fluorophore (e.g., for in vivo applications). In certain embodiments, the mesoscale nanoparticle composition comprises a radiolabel noncovalently associated with the nanoparticle and/or attached to the nanoparticle.
In another aspect, the invention is directed to a method of making the mesoscale nanoparticle composition, the method comprising: introducing a first solution into a second solution in a drop-wise manner while stirring (or otherwise mixing or agitating) the second solution, wherein the first solution comprises the core in a solvent (e.g., acetonitrile, DMSO) (e.g., at a concentration from about 1 mg/ml to about 100 mg/ml, e.g., about 10 mg/ml to about 75 mg/ml, e.g., about 50 mg/ml).
In another aspect, the invention is directed to a method of treating a patient, the method comprising administering the mesoscale nanoparticle composition to the patient suffering from or susceptible to a disease or condition affecting the kidney (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, clear cell renal cell carcinoma).
In certain embodiments, the disease or condition is a member selected from the group consisting of renal carcinoma, acute kidney disease, chronic kidney disease, heart failure, liver cirrhosis, hypertension, and renal failure.
In another aspect, the invention is directed to a method for monitoring a patient, the method comprising administering the mesoscale nanoparticle composition for investigating renal blood flow or renal physiology.
In certain embodiments, the method comprises imaging the administered mesoscale nanoparticle composition.
In certain embodiments, the administered mesoscale nanoparticle composition demonstrates selective targeting of kidneys of the patient such that concentration of the nanoparticle in the kidneys is at least 1.5 times greater than concentration of the nanoparticle in any of the heart, lung, liver, or spleen of the patient (e.g., at least 1.5 times greater, at least 2 times greater, at least 3 times greater, 4 times greater, at least 5 times greater, at least 6 times greater, or at least 7 times greater) at a given time following administration of the composition, wherein the given time is from 3 days to 2 months following administration.
In another aspect, the invention is directed to a mesoscale nanoparticle composition (e.g., a pharmaceutical composition) comprising: a core (e.g., polymer, poly(lactic-co-glycolic acid) (PLGA), silica, chitosan, lipid, liposome, metal e.g., gold, iron oxide); and a surface coating (e.g., polyethylene glycol, non-opsonizing lipid) having a surface charge between −40 mV to +40 mV (or −25 mV to +25 mV, or −15 mV to +15 mV), wherein the composition is in the form of a nanoparticle having a diameter (e.g.; average diameter) from 200 nm to 1000 nm (or 250 nm to 500 nm) (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) for use in a method of treating a disease or condition affecting a kidney (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, clear cell renal cell carcinoma) in a subject (e.g., suffering from or susceptible to a disease, disorder, or condition), wherein the treating comprises delivering the composition to the kidney in the subject.
In another aspect, the invention is directed to a mesoscale nanoparticle composition (e.g., a pharmaceutical composition) comprising: a core (e.g., polymer, poly(lactic-co-glycolic acid) (PLGA), silica, chitosan, lipid, liposome, metal e.g., gold, iron oxide); and a surface coating (e.g., polyethylene glycol, non-opsonizing lipid) having a surface charge between −40 mV to +40 mV (or −25 mV to +25 mV, or −15 mV to +15 mV), wherein the composition is in the form of a nanoparticle having a diameter (e.g.; average diameter) from 200 nm to 1000 nm (or 250 nm to 500 nm) (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) for use in a method of in vivo diagnosis of a disease or condition affecting a kidney (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, clear cell renal cell carcinoma) in a subject (e.g., suffering from or susceptible to a disease, disorder, or condition), wherein the in vivo diagnosis comprises delivering the composition to the kidney in the subject.
In another aspect, the invention is directed to a mesoscale nanoparticle composition (e.g., a pharmaceutical composition) comprising: a core (e.g., polymer, poly(lactic-co-glycolic acid) (PLGA), silica, chitosan, lipid, liposome, metal e.g., gold, iron oxide); and a surface coating (e.g., polyethylene glycol, non-opsonizing lipid) having a surface charge between −40 mV to +40 mV (or −25 mV to +25 mV, or −15 mV to +15 mV), wherein the composition is in the form of a nanoparticle having a diameter (e.g.; average diameter) from 200 nm to 1000 nm (or 250 nm to 500 nm) (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) for use in (a) a method of treating a disease or condition affecting a kidney (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, clear cell renal cell carcinoma) in a subject (e.g., suffering from or susceptible to a disease, disorder, or condition), or (b) a method of in vivo diagnosis of a disease or condition affecting a kidney (e.g., acute kidney injury, chronic kidney disease, polycystic kidney disease, clear cell renal cell carcinoma) in a subject (e.g., suffering from or susceptible to a disease, disorder, or condition), wherein the method comprises delivering the composition to the kidney in the subject.
In another aspect, the invention is directed to a mesoscale nanoparticle composition (e.g., a pharmaceutical composition) comprising: a core (e.g., polymer, poly(lactic-co-glycolic acid) (PLGA), silica, chitosan, lipid, liposome, metal e.g., gold, iron oxide); and a surface coating (e.g., polyethylene glycol, non-opsonizing lipid) having a surface charge between −40 mV to +40 mV (or −25 mV to +25 mV, or −15 mV to +15 mV), wherein the composition is in the form of a nanoparticle having a diameter (e.g.; average diameter) from 200 nm to 1000 nm (or 250 nm to 500 nm) (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) for use in therapy.
In another aspect, the invention is directed to a mesoscale nanoparticle composition (e.g., a pharmaceutical composition) comprising: a core (e.g., polymer, poly(lactic-co-glycolic acid) (PLGA), silica, chitosan, lipid, liposome, metal e.g., gold, iron oxide); and a surface coating (e.g., polyethylene glycol, non-opsonizing lipid) having a surface charge between −40 mV to +40 mV (or −25 mV to +25 mV, or −15 mV to +15 mV), wherein the composition is in the form of a nanoparticle having a diameter (e.g.; average diameter) from 200 nm to 1000 nm (or 250 nm to 500 nm) (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) for use in in vivo diagnosis.
In certain embodiments, the delivered mesoscale nanoparticle composition can be imaged.
In certain embodiments, the core comprises PLGA. In certain embodiments, a base of PLGA is modified. In certain embodiments, the PLGA has a molecular weight from 7 kDa to 54 kDa. In certain embodiments, the surface coating (e.g., polyethylene glycol, non-opsonizing lipid) has a molecular weight from 2 kDa to 10 kDa (e.g., from 4 kDa to 7 kDa, e.g., 5 kDa). In certain embodiments, the surface coating is selected from the group consisting of polyethylene glycol (PEG), PEG-carboxylic acid, PEG-carboxylic acid-DMAB, and methoxy PEG. In certain embodiments, the weight ratio of PEG to PLGA is from 9% to 13% (e.g., prior to synthesis of the mesoscale nanoparticle composition).
In certain embodiments, the mesoscale nanoparticle composition comprises a therapeutic agent (e.g., a hydrophobic small molecule; a targeted chemotherapeutic (e.g., doxorubicin, sorafenib, or sunitinib); a metabolic targeting therapeutic (e.g., STF-31, CPI-613, or Fasentin); alpha-7 nicotine receptor antagonist; a hypertension therapeutic; a TGFbeta inhibitor (e.g., kinase inhibitors (e.g., LY2157299 (galunisertib), SD-208, SB505124)); a reactive oxygen species and DNA damage inhibitor (e.g., Amifostine (Ethyol)), a Nf kappa B inhibitor (e.g., Pyrrolidine dithiocarbamate, quinazoline, BMS-345541, BAY-11-7085); a p21 inhibitor (e.g., siRNA or small-molecule inhibitor); a mTOR inhibitor (e.g., RAD001 (everolimus)); a glutaminase inhibitor (e.g., BPTES, CB-839); a therapeutic as listed in Table 3; and/or a diuretic).
In certain embodiments, the therapeutic agent is noncovalently associated with the nanoparticle, e.g., encapsulated within the surface coating.
In certain embodiments, the therapeutic agent is attached to the nanoparticle (e.g., non-covalently attached or covalently bound to a moiety of the surface coating).
In certain embodiments, the mesoscale nanoparticle composition comprises an imaging agent (e.g., a dye, e.g., a fluorescent molecule, e.g., a fluorescent molecule within the core). In certain embodiments, the imaging agent is selected from the group consisting of a near infrared dye (e.g., 3,3′-Diethylthiadicarbocyanine iodide) and a far-red fluorophore (e.g., for in vivo applications). In certain embodiments, the mesoscale nanoparticle composition comprises a radiolabel noncovalently associated with the nanoparticle and/or attached to the nanoparticle.
In certain embodiments, the disease or condition is a member selected from the group consisting of renal carcinoma, acute kidney disease, chronic kidney disease, heart failure, liver cirrhosis, hypertension, and renal failure.
In certain embodiments, the mesoscale nanoparticle composition can be used in a method of investigating renal blood flow or renal physiology.
In certain embodiments, the mesoscale nanoparticle composition demonstrates selective targeting of the kidney of the subject such that concentration of the nanoparticle in the kidneys is at least 1.5 times greater than concentration of the nanoparticle in any of the heart, lung, liver, or spleen of the patient (e.g., at least 1.5 times greater, at least 2 times greater, at least 3 times greater, 4 times greater, at least 5 times greater, at least 6 times greater, or at least 7 times greater) at a given time following administration of the composition, wherein the given time is from 3 days to 2 months following administration.
Elements of embodiments involving one aspect of the invention (e.g., methods) can be applied in embodiments involving other aspects of the invention (e.g., systems), and vice versa.
DefinitionsIn order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.
In this application, the use of “or” means “and/or” unless stated otherwise. As used in this application, the term “comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps. As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
“Administration”: The term “administration” refers to introducing a substance into a subject. In general, any route of administration may be utilized including, for example, parenteral (e.g., intravenous), oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments. In some embodiments, administration is oral. Additionally or alternatively, in some embodiments, administration is parenteral. In some embodiments, administration is intravenous.
“Associated”: As used herein, the term “associated” typically refers to two or more entities in physical proximity with one another, either directly or indirectly (e.g., via one or more additional entities that serve as a linking agent), to form a structure that is sufficiently stable so that the entities remain in physical proximity under relevant conditions, e.g., physiological conditions. In some embodiments, associated moieties are covalently linked to one another. In some embodiments, associated entities are non-covalently linked. In some embodiments, associated entities are linked to one another by specific non-covalent interactions (e.g., by interactions between interacting ligands that discriminate between their interaction partner and other entities present in the context of use, such as, for example. streptavidin/avidin interactions, antibody/antigen interactions, etc.). Alternatively or additionally, a sufficient number of weaker non-covalent interactions can provide sufficient stability for moieties to remain associated. Exemplary non-covalent interactions include, but are not limited to, electrostatic interactions, hydrogen bonding, affinity, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, pi stacking interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, etc.
“Biocompatible”: The term “biocompatible”, as used herein is intended to describe materials that do not elicit a substantial detrimental response in vivo. In certain embodiments, the materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and/or their administration in vivo does not induce inflammation or other such adverse effects. In certain embodiments, materials are biodegradable.
“Biodegradable”: As used herein, “biodegradable” materials are those that, when introduced into cells, are broken down by cellular machinery (e.g., enzymatic degradation) or by hydrolysis into components that cells can either reuse or dispose of without significant toxic effects on the cells. In certain embodiments, components generated by breakdown of a biodegradable material do not induce inflammation and/or other adverse effects in vivo. In some embodiments, biodegradable materials are enzymatically broken down. Alternatively or additionally, in some embodiments, biodegradable materials are broken down by hydrolysis. In some embodiments, biodegradable polymeric materials break down into their component polymers. In some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymeric materials) includes hydrolysis of ester bonds. In some embodiments, breakdown of materials (including, for example, biodegradable polymeric materials) includes cleavage of urethane linkages.
“Carrier”: As used herein, “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
“Substantially”: As used herein, the term “substantially”, and grammatic equivalents, refer to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
“Subject”: As used herein, the term “subject” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are be mammals, particularly primates, especially humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.
“Therapeutic agent”: As used herein, the phrase “therapeutic agent” refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject.
“Treatment”: As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a substance that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
Drawings are presented herein for illustration purposes, not for limitation.
The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and better understood by referring to the following description taken in conduction with the accompanying drawings, in which:
Throughout the description, where compositions are described as having, including, or comprising specific components, or where methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.
The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.
Described herein are synthesized “mesoscale” nanoparticles (“MNPs”), approximately 400 nm in diameter, which unexpectedly localized selectively in renal proximal tubules up at least 1.5 times more efficiently in the kidney than other organs. Furthermore, the nanoparticles described herein differ from those previously described primarily in size, PLGA size, PEG size, PEG functionality, and charge. Although nanoparticles typically localize in the liver and spleen, modulating their size and opsonization potential allowed for stable targeting of the kidneys through a new proposed uptake mechanism. Applying this kidney targeting strategy enables use in the treatment of renal disease and the study of renal physiology.
Imaging AgentsIn certain embodiments, the compositions described herein include (i) imaging agents that are, or are associated with, the therapeutic agent, and/or (ii) imaging agents that are associated with, or are a part of, the MNPs. In certain embodiments, the imaging agents can include radiolabel s, radionuclides, radioisotopes, fluorophores, fluorochromes, dyes, metal lanthanides, paramagnetic metal ions, superparamagnetic metal oxides, ultrasound reporters, x-ray reporters, and/or fluorescent proteins.
In certain embodiments, radiolabels comprise 99mTc, 64Cu, 67Ga, 186Re, 188Re, 153Sm, 177Lu, 67Cu, 123I, 124I, 125I, 11C, 13C, 15O, 18F, 186Re, 188Re, 153Sm, 166Ho, 177Lu, 149Pm, 90Y, 212Bi, 103Pd, 109Pd, 159Gd, 140La, 198Au, 199Au, 169Yb , 175Yb, 165Dy, 166Dy, 67Cu, 105Rh, 111Ag, 89Zr, and 192Ir. In some embodiments, paramagnetic metal ions comprise Gd(III), Dy(III), Fe(III), and Mn(II). In some embodiments, Gadolinium (III) contrast agents comprise Dotarem, Gadavist, Magnevist, Omniscan, OptiMARK, and Prohance. In certain embodiments, x-ray reporters comprise iodinated organic molecules or chelates of heavy metal ions of atomic numbers 57 to 83.
In certain embodiments, PET (Positron Emission Tomography) tracers are used as imaging agents. In some embodiments, PET tracers comprise 89Zr, 64Cu, [18F] fluorodeoxyglucose.
In certain embodiments, fluorophores comprise fluorochromes, fluorochrome quencher molecules, any organic or inorganic dyes, metal chelates, or any fluorescent enzyme substrates, including protease activatable enzyme substrates. In some embodiments, fluorophores comprise long chain carbophilic cyanines. In other embodiments, fluorophores comprise DiI, DiR, DiD, and the like. Fluorochromes comprise far red, and near infrared fluorochromes (NIRF). Fluorochromes include but are not limited to a carbocyanine and indocyanine fluorochromes. In some embodiments, imaging agents comprise commercially available fluorochromes including, but not limited to Cy5.5, Cy5 and Cy7 (GE Healthcare); AlexaFlour660, AlexaFlour680, AlexaFluor750, and AlexaFluor790 (Invitrogen); VivoTag680, VivoTag-S680, and VivoTag-S750 (VisEn Medical); Dy677, Dy682, Dy752 and Dy780 (Dyomics); DyLight547, DyLight647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and Kodak X-SIGHT 650, Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Carestream Health).
AdministrationPharmaceutical compositions incorporating the MPs described herein may be administered according to any appropriate route and regimen. In some embodiments, a route or regimen is one that has been correlated with a positive therapeutic benefit.
In certain embodiments, the exact amount administered may vary from subject to subject, depending on one or more factors as is well known in the medical arts. Such factors may include, for example, one or more of species, age, general condition of the subject, the particular composition to be administered, its mode of administration, its mode of activity, the severity of disease; the activity of the specific MNP employed; the specific pharmaceutical composition administered; the half-life of the composition after administration; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and the like. Pharmaceutical compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions will be decided by an attending physician within the scope of sound medical judgment.
Compositions described herein may be administered by any route, as will be appreciated by those skilled in the art. In certain embodiments, compositions described herein are administered by oral (PO), intravenous (IV), intramuscular (IM), intra-arterial, intramedullary, intrathecal, subcutaneous (SQ), intraventricular, transdermal, interdermal, intradermal, rectal (PR), vaginal, intraperitoneal (IP), intragastric (IG), topical (e.g., by powders, ointments, creams, gels, lotions, and/or drops), mucosal, intranasal, buccal, enteral, vitreal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter.
In some embodiments, the pharmaceutical compositions and/or MNPs thereof may be administered intravenously (e.g., by intravenous infusion), by intramuscular injection, by intratumoural injection, and/or via portal vein catheter, for example. However, the subject matter described herein encompasses the delivery of pharmaceutical compositions and/or MNPs thereof in accordance with embodiments described herein by any appropriate route taking into consideration likely advances in the sciences of drug delivery.
To investigate the parameters that may influence nanoparticle localization, the literature was probed to construct a simple plot to consolidate nanoparticle localization data from multiple studies as shown in
It is found herein that nanomaterial size has a demonstrated effect on biodistribution. Certain synthetic polymers, low-molecular weight proteins, and peptides less than 20 kDa in molecular weight exhibit renal tubule biodistribution but are quickly cleared from the body. Nanoparticles less than 250 nm tend to accumulate in the liver or spleen, either through MPS trafficking or entrance through liver fenestrations (approximately 100 nm) as shown in
Herein, mesoscale nanoparticles (MNPs) were synthesized to avoid MPS organs and to selectively and stably accumulate in the kidneys up to seven times more efficiently than other organs. The parameter space required for this localization in terms of particle size and opsonization potential was determined. The nanoparticles accumulated in proximal versus distal renal tubules and more so at their basal rather than the apical membranes. Without being bound by theory, this specific accumulation may be due to a mechanism in which MNPs are endocytosed by endothelial cells of the peritubular capillaries because of the pressure drop in the nephron and the large absorptive pressure of the capillaries. Applying this targeting strategy, mesoscale nanoparticles can be used to treat diseases that affect the proximal tubules of the kidneys and to study renal blood flow.
Anionic (A-MNPs) and cationic (C-MNPs) coated with poly(lactic-co-glycolic acid) (PLGA) functionalized with PEG were synthesized and measured to be approximately 400 nm in diameter. The nanoparticles were loaded with a fluorescent dye for biodistribution studies. Carboxylic acid-terminated PLGA was conjugated to heterobifunctional amine-PEG-carboxylic acid. 1H NMR was performed to confirm conjugation. The nanoprecipitation method was used to form A-MNPs of 386.7 nm in diameter with a potential of −19.5 mV as determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) and is shown in Table 1,
In order to explore nanoparticle stability, aggregation, dye release, and potential assays were performed. Each particle formulation exhibited similar stability after incubation in PBS for three days as shown by a particle size as shown in
Table 1 shows size (DLS) and surface charge (zeta potential) data of dye-loaded mesoscale nanoparticles.
The nanoparticles selectively accumulated in the kidneys of mice following intravenous injection. 50 mg/kg of A- or C-MNPs was intravenously injected into female SKH-1 mice and imaged the animals daily for up to 7 days and bi-weekly thereafter for approximately 3 months. Biodistribution was measured by fluorescence in vivo in order to track nanoparticle localization and degradation over time, a widely-used method that closely approximates other biodistribution assays.
Neither mice treated with anionic (A-MNP) nor cationic mesoscale nanoparticles (C-MNPs) exhibited a significant weight change at day 3 or day 7 compared to dye alone-treated mice after 3 days as shown in
Nanoparticles localized to both kidneys and the chest region as shown in
Upon organ extraction, to obtain more quantitative biodistribution patterns, the fluorescence signal was significantly greater in the kidneys than in any other organ analyzed as shown in
The biodistribution of neutral (ζ potential=0.38 mV) mesoscale nanoparticles (N-MNPs), functionalized with methoxy-PEG (mPEG), was also measured and is shown in
As shown in
Opsonizing PLGA nanoparticles without PEG (O-MNPs), loaded with IR783 dye, localized in the liver 30 minutes following injection as shown in
Fluorescence imaging in live mice underestimates signal in the kidneys, heart, and other dense tissues compared to ex vivo quantification.
The difference in nanoparticle fluorescence emission intensity between organs in vivo and ex vivo was assessed. One mouse, treated with 50 mg/kg A-MNPs, was euthanized 2 days following injection. In vivo, prior to euthanization, the fluorescence localization pattern was identical to that shown in similar experiments with the same treatment and is shown in FIG. 3A and
In order to provide higher order spatial information regarding the distribution of nanoparticles in the kidney, both immunofluorescence (IF) and immunohistochemistry (IHC) techniques was used. Kidney tissue from treated or control mice was sectioned and stained for CD31 (blood vessels) or E-cadherin (distal tubules) expression by IF and for the presence of PEG by IHC. Nanoparticle fluorescence in the renal tubules of MNP-treated mice was significantly higher than negative controls as shown in
The parameters necessary to effect localization toward the kidneys and away from other organs such as the liver and spleen were investigated. The role of nanoparticle size with respect to the organ of localization is not clear. Also, it is apparent from this study that a relatively small surface charge, or lack thereof, does not significantly affect biodistribution because A-MNPs, C-MNPs, and N-MNPs all localized in the kidneys. These findings, in agreement with the literature as shown in
Next, the mechanism of localization to the kidney at the tissue level was probed. Histology confirmed that MNPs predominantly localized in the basolateral region of proximal tubule epithelial cells. Previous work showed that nanoparticles of approximately 80 nm in diameter with a non-opsonizing surface targeted the kidney glomeruli. The fenestrations of this segment of the nephron (approximately 80-100 nm) are too small for the MNPs described herein to pass through, however. There are also fenestrations in the peritubular capillaries which run along the renal tubules; however those are also reportedly too small (˜5 nm) for passage of MNPs. Thus, MNPs must be endocytosed by endothelial cells of the peritubular capillaries, which is confirmed in vitro and depicted in
The present disclosure describes synthesis and characterization of a class of polymeric mesoscale nanoparticles which selectively and stably localized in the proximal tubule epithelium of the kidneys. Exploring the parameter spaces responsible for kidney targeting revealed that low opsonization potential is important for kidney targeting, but moderate changes in zeta potential had no effect on localization. This data suggests a mechanism of localization to the proximal tubules supported by histological evidence whereby the nanoparticles are endocytosed by endothelial cells of the peritubular capillaries because of the pressure drop in the nephron and the large absorptive pressure of the capillaries. This targeting strategy can be applied to the treatment of diseases which affect the proximal tubules and as a tool for studying renal physiology.
Experimental Examples Nanoparticle Biodistribution Literature SurveyA brief literature survey was conducted to investigate the effects of nanoparticle size and opsonization potential on biodistribution. Publications that studied nanoparticle biodistribution without the use of molecular targeting moieties were selected. Studies performed in diseased animals were excluded in order to determine biodistribution in healthy, uncompromised mice or rats. Only nanoparticles administered intravenously were included. The nanoparticle diameter, surface functionalization, and primary site of localization were recorded from each paper. Of publications disclosing many nanoparticles with minor size iterations, one representative particle was chosen. The opsonization potential of each particle was assigned a score with 1 being the most opsonizing and 5 being the least opsonizing.
Category binning was performed as follows: 1—PLGA or gold particles with no coating; 2—Relatively opsonizing proteins, small molecule labels, or polymers; 3—Relatively non-opsonizing protein or polymer (as described in the original manuscript); 4—PEG-coated particles with surface charge −15<X>+15; 5—PEG-coated particles with surface charge −15>X<+15 or non-opsonizing lipid. Each cited nanoparticle was assigned a number as shown in Table 2. The size and assigned degree of opsonization (e.g., surface category) were plotted for each referenced nanoparticle as shown in
Table 2 shows an exemplary nanoparticle distribution literature survey.
Poly(lactic-co-glycolic acid) conjugated to polyethyle glycol (PLGA-PEG) was synthesized similarly to previously described methods as described by Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad, O. C. Biomaterials 2007, 28, 869-876. Carboxylic acid-terminated poly(lactic-co-glycolic acid) (PLGA) (50:50; MW 38-54 kDa) (90-130 μmol) (Aldrich; St. Louis, Mo.) was dissolved in 10 mL methylene chloride and activated with N-hydroxysuccinimide (NHS, 135 mg, 1.2 mmol) (Aldrich) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, 230 mg, 1.2 mmol) for 30 minutes with stirring. Conjugated PLGA-NHS was precipitated with 5 mL ethyl ether, washed 3× with cold 50:50 ethyl ether:methanol, and dried under vacuum. To PLGA-NHS (1 g, 18-26 μmol) in 4 mL chloroform, 250 mg (50 μmol) NH2-PEG-COOH (MW 5 kDa) (Nanocs; New York, N.Y.) was added with 28 mg (220 μmop N,N-diisopropylethylamine. After the reaction proceeded with mixing overnight, conjugated PLGA-PEG was precipitated and washed 3× with 5 mL cold methanol and allowed to dry under vacuum. The polymer was characterized by 1H NMR as previously described in Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad, 0. C. Biomaterials 2007, 28, 869-876.
PLGA-PEG was used to form anionic mesoscale nanoparticles (A-MNPs) by the nanoprecipitation method. PLGA-PEG (100 mg) was dissolved with the fluorescent Cy5 mimic 3,3′-Diethylthiadicarbocyanine iodide (DEDC) (Acros Organics; Geel, Belgium) (10 mg) in 2 mL acetonitrile. This solution was added dropwise to 4 mL water with 100 μL 10% Pluronic F-68 (Gibco; Grand Island, N.Y.) and stirred for 2 hours. The solution was then centrifuged for 15 minutes at 6,600 RPM, washed, and centrifuged again. Particles were lyophilized in a 2% sucrose solution for storage at −20° C. Dried particles were suspended in phosphate-buffered saline (PBS) or water and analyzed for size by dynamic light scattering (DLS) (Malvern; Worcestershire, United Kingdom) and scanning electron microscopy (SEM) following gold-palladium coating (Zeiss; Oberkochen, Germany), for potential by electrophoretic light scattering (ELS) (Malvern), and for encapsulation by UV-VIS absorbance (Jasco; Easton, Md.) and DEDC fluorescence (Tecan; Mannedorf, Switzerland). To synthesize cationic mesoscale nanoparticles (C-MNPs), 9.5 mg lyophilized A-MNPs were suspended in a solution of 0.5 mg/mL didodecyldimethylammonium bromide (DMAB). This method can modify an anionic PLGA-PEG nanoparticle surface, resulting in cationic nanoparticles functionally shown to be stable for in vitro and in vivo applications. Before use, both A-MNPs and C-MNPs were centrifuged and resuspended in PBS to remove unincorporated dye and/or DMAB.
All experiments performed in animals were approved by and carried out in accordance with the MSKCC Institutional Animal Care and Use Committee. Female 4-8 week SKH-1 Elite hairless mice (Crl:SKH1-Hrhr) (Charles River; Troy, N.Y.) were used in order to reduce background autofluorescence or absorbance from haired mice. They were fed irradiated 5V75 alfalfa-free food (LabDiet; St. Louis, Mo.) to reduce fluorescent background in imaging. Groups of 6 mice each were injected intravenously via the tail vein with 50 mg/kg of A-MNPs or C-MNPs encapsulating DEDC. Control groups of 4 mice each were injected with 100 μL PBS or 23 μg/kg DEDC in PBS with 0.5% DMSO (equal to the amount of encapsulated dye in particle-injected mice). Mice were imaged dorsally with an IVIS Spectrum Pre-clinical In vivo Imaging System (Perkin Elmer; Waltham, Mass.) using 640 nm excitation and 680 nm emission filters to determine fluorescence biodistribution at the following times post-injection: 30 minutes, 4 hours, and 1-7 days. In vivo fluorescence images were analyzed using Living Image Software v4.3 (Perkin Elmer) with regions of interest (ROIs) selected around each kidney and the central lung region to obtain total fluorescence efficiency (TFE) from each. On day 3, 3 mice from the A-MNP and C-MNP groups, 2 mice from the PBS control group, and all 4 mice from the DEDC control group were euthanized by carbon dioxide overdose. The following organs were harvested and imaged for fluorescence: heart, lungs, kidneys, liver, and spleen. TFE for each organ was obtained and normalized by organ weight to obtain organ-level biodistribution. Normalized organ fluorescence for each group was averaged and standard deviations obtained. The organs were weighed and fixed with 4% paraformaldehyde (PFA) overnight at 4° C. One mouse treated with anionic nanoparticles was imaged by fluorescence and X-ray computed tomography (CT) with an IVIS Spectrum CT (Perkin Elmer) one day following injection. Three-dimensional reconstruction of fluorescent foci around the kidneys was performed with multiple imaging fields and overlaid onto a computed tomographic image of the mouse in order to confirm kidney localization and determine particle distribution throughout the organ.
Fixed organs were dehydrated and paraffin embedded before 5 μm sections were placed on glass slides. The paraffin was removed and the slides were stained with haematoxylin and eosin (H&E) for basic histology. Another set of slides for immunofluorescence were stained with 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei of cells and either an anti-CD31 antibody to stain endothelial cells (Dianova, Cat # DIA-310) with concentration of 1 μs/ml or anti-E-cadherin (BD Bioscience; San Jose, Calif.; Cat #610181) with concentration of 5 μg/ml to stain epithelial cells. For detection, isotype-specific secondary antibodies conjugated to AlexaFluor 488 (Invitrogen; Carlsbad, Calif.; Cat # T20922) were used at 1:1000 dilution according to manufacturer's instruction. Immunohistochemistry was performed using a Discovery XT processor (Ventana Medical Systems; Tucson, Ariz.). The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems) and sections were blocked for 30 minutes with 10% normal rabbit serum in PBS+0.1% BSA. Anti-PEG (Abcam; Cambridge, Mass.; Cat # ab94764, 5 ug/ml) antibodies specific to the PEG backbone were applied and sections were incubated for 5 hours, followed by a 60 minute incubation with biotinylated rabbit anti-rat IgG (Vector Labs; Burlingame, Calif.; Cat # PK-4004) at 1:200 dilution. The assay was performed with a DAB detection kit (Ventana Medical Systems) according to manufacturer instructions. Slides were counterstained with hematoxylin (Ventana Medical Systems) and coverslipped with Permount (Fisher Scientific; Hampton, N.H.).
Slides were imaged with an Olympus IX51 inverted light microscope with slide adapter (Olympus; Center Valley, Pa.) outfitted with an Olympus DP73 digital color camera and Olympus XM10 monochrome camera. Fluorescent slides were excited with filtered light from a X-Cite 120Q lamp (Lumen Dynamics; Ontario, Canada). Fluorescence images were acquired with appropriate filter cubes for DAPI, AlexaFluor 488, and Cy5 with constant exposure times for each fluorophore and analyzed in Image? (National Institutes of Health; Bethesda, Md.) with constant brightness values for each.
Nanoparticle Toxicity StudyMice treated with A- or C-MNPs, dye alone, or PBS alone were weighed immediately preceding injection, on the third day following treatment, and on the seventh day following treatment. The kidneys from mice in each group were sectioned as described herein. Sections were reviewed by a licensed anatomic pathologist for histomorphological evidence of damage.
Mice in each group were imaged using an IVIS Spectrum Pre-Clinical In vivo Imaging System for 7 days as described in the main Materials and Methods. Separately, 3 mice, comprising a mouse treated with PBS alone, A-MNPs, or C-MNPs were imaged every 1-2 weeks for approximately 3 months following injection to measure long-term fluorescence attenuation, which is interpreted to signify MNP degradation.
PLGA-methoxy PEG Nanoparticle CharacterizationNeutral PLGA-mPEG mesoscale nanoparticles (N-MNPs) were synthesized and characterized essentially identically to PLGA nanoparticles functionalized with NH2—PEG-COOH, which was described in the main Materials and Methods. Instead of NH2—PEG-COOH, however, NH2—PEG-methoxy (MW 5 kDa) (Nanocs; New York, N.Y.) was conjugated to carboxylic acid-terminated PLGA. The polymer was characterized by 1H NMR. Nanoparticles encapsulating DEDC were formed with this polymer and characterized for size, potential, and encapsulation as described in the main manuscript.
A group of 3 SKH-1 Elite hairless mice were fed with 5V75 chow for at least 1 week before the experiment to reduce fluorescent background from alfalfa-containing food. Mice were injected intravenously via the tail vein with 50 mg/kg of N-MNPs encapsulating DEDC for fluorescence localization studies. One control mouse was injected with 125 μL PBS. These mice were imaged 30 minutes, 3 hours, and 1-3 days following injection with an IVIS Spectrum using 640 nm excitation and 680 nm emission filters. Total combined fluorescence efficiency from both kidneys was measured. At day 3, the mice were euthanized and the following organs were recovered, imaged, and weighed: kidneys, heart, lungs, spleen, and liver.
PLGA Nanoparticle CharacterizationOpsonizing PLGA mesoscale nanoparticles (O-MNPs) were synthesized similarly to PLGA-PEG and PLGA-mPEG nanoparticles as described in the main Materials and Methods. Nanoparticles were formed by nanoprecipiation with PLGA and IR-783 fluorescent dye (25 mg used). The size, potential, and encapsulation efficiency were measured. It should be noted that IR-783 was used instead of DEDC due to precipitate formation with the latter.
A group of 8 SKH-1 Elite hairless mice fed with 5V75 food were injected intravenously via the tail vein with 50 mg/kg of O-MNPs encapsulating IR-783 for fluorescence localization studies. Two control mice were injected with 125 μL PBS. These mice were imaged 30 minutes, 4 hours, and 24 hours following injection with the 647 nm excitation and 820 nm emission filters of an IVIS Spectrum. Four treated mice and one control mouse were euthanized after 24 hours. The remaining mice were imaged at 2 and 3 days following injection before euthanization. Following euthanization, the kidneys, heart, lungs, spleen, and liver were recovered, imaged, and weighed. Total fluorescence efficiency normalized by weight of each organ was obtained.
Ex Vivo to In Vivo ComparisonA SKH-1 Elite hairless mouse injected with 50 mg/kg A-MNPs was imaged via an IVIS Spectrum using the 650 nm excitation and 680 nm emission filters 2 days following injection. The mouse was euthanized and kidneys, heart, lungs, spleen, and liver were extracted, leaving other organs intact and in place. The mouse without organs was then imaged as well as the organs alone.
To determine the difference in fluorescence from kidneys ex vivo versus in vivo, regions of interest (ROIs) were drawn around the kidneys and central lung region and TFE was obtained for each ROI. After euthanization and organ imaging, TFE was obtained for both kidneys and lungs. TFE for kidneys ex vivo was divided by that of kidneys in vivo to obtain the in vivo underestimation ratio. The same method was performed for the lungs.
The difference in nanoparticle fluorescence emission intensity between organs in vivo and ex vivo was assessed. One mouse, treated with 50 mg/kg A-MNPs, was euthanized 2 days following injection. In vivo, prior to euthanization, the fluorescence localization pattern was identical to that shown in similar experiments with the same treatment as shown in
The following cell lines were used to determine uptake of anionic and cationic nanoparticles in vitro: MCF-7 human breast adenocarcinoma (ATCC; Manassas, Va.), SK-RC-48 human clear cell renal cell carcinoma (ccRCC) (Weill Cornell Medical College; New York, N.Y.), bEND3 mouse brain endothelial (ATCC), endothelial cell line EA-926, and human kidney proximal tubular epithelial cell line HK-2. MCF-7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with penicillin (10,000 U/mL)/streptomycin (10,000 U/mL)/glutamine (29.2 mg/mL) (Gibco; Carlsbad, Calif.), 0.01 mg/mL human recombinant insulin (Gibco), and 10% fetal bovine serum (FBS) (Gibco); SK-RC-48 were cultured in DMEM with 1× penicillin/streptomycin/glutamine and 10% FBS; and bEND3 cells in the same plus 1% glutaMAX (Gibco). HK-2 cells were cultured in Keratinocyte Serum Free Media (Gibco). EA-926 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS. Cells were passaged approximately weekly and media changed every 2-3 days. Cells were seeded into a 6-well cell culture dish for uptake studies. A-MNPs and C-MNPs in PBS were diluted in the appropriate medium at a final concentration of 100 μg/mL and incubated with each cell line for 10 minutes. Cells were washed with 2×1 mL PBS before adding fresh media. Fluorescent images were captured with the microscope and camera setup described in the main Materials and Methods section with Cy5 filters 10 minutes following washing. Images were obtained with identical exposure times and processed in ImageJ with identical brightness settings.
Table 3 shows exemplary disease targets, therapeutics (e.g., classes of drugs/inhibitors), and a brief rationale. In certain embodiments, therapeutics can be encapsulated within the MNPs.
Because of the kidney-targeting ability of the drug carrier nanoparticles, treatment of the kidney can now be performed using therapeutic agents that are currently not used for kidney treatment and/or that are currently experimental. Various therapeutic agents (e.g., TGFb inhibitors, mTOR inhibitors, everolimus, kinase inhibitors) have not been usable for kidney disease treatment due to poor pharmacokinetics. By the time the kidneys respond to a delivered therapeutic agent, toxicity side-effects occur elsewhere in the body. Accordingly, the mesoscale nanoparticles disclosed herein can selectively deliver therapeutic agents (e.g., therapeutic agents that are typically not used for kidney disease treatment due to poor pharmacokinetics) to the kidney while minimizing toxicity elsewhere in the body.
Claims
1. A mesoscale nanoparticle composition comprising:
- a core comprising poly(lactic-co-glycolic acid) (PLGA); and
- a surface coating comprising polyethylene glycol having a surface charge between −40 mV to +40 mV,
- wherein the composition is in the form of a nanoparticle having an intensity-weighted average diameter as determined by dynamic light scattering from 200 nm to 500 nm; and
- at least one of a therapeutic agent, a positron emission tomography (PET) tracer, a dye, and a fluorescent molecule associated with the nanoparticle, wherein the therapeutic agent comprises a hydrophobic small molecule, a targeted chemotherapeutic, a metabolic targeting therapeutic, an alpha-7 nicotine receptor antagonist, a hypertension therapeutic; a TGFbeta inhibitor, a reactive oxygen species and DNA damage inhibitor, a Nf kappa B inhibitor, a p21 inhibitor; a mTOR inhibitor, or a glutaminase inhibitor.
2. (canceled)
3. The mesoscale nanoparticle composition of claim 1, wherein a base of PLGA is modified.
4. The mesoscale nanoparticle composition of claim 1, wherein the PLGA has a molecular weight from 7 kDa to 54 kDa.
5. The mesoscale nanoparticle composition of claim 1, wherein the surface coating has a molecular weight from 2 kDa to 10 kDa.
6. The mesoscale nanoparticle composition of claim 1, wherein the surface coating is selected from the group consisting of polyethylene glycol (PEG), PEGcarboxylic acid, PEG-carboxylic acid-DMAB, and methoxy PEG.
7. The mesoscale nanoparticle composition of claim 6, wherein the weight ratio of PEG to PLGA is from 9% to 13%.
8. (canceled)
9. The mesoscale nanoparticle composition of claim 1, wherein the therapeutic agent is noncovalently associated with the nanoparticle, e.g., encapsulated within the surface coating.
10-12. (canceled)
13. The mesoscale nanoparticle composition of claim 1, wherein a radiolabel is noncovalently associated with the nanoparticle.
14. (canceled)
15. A method of treating a patient, the method comprising administering the mesoscale nanoparticle composition of claim 1 to the patient suffering from or susceptible to a disease or condition affecting the kidney.
16. The method of claim 15, wherein the disease or condition is a member selected from the group consisting of renal carcinoma, acute kidney disease, chronic kidney disease, heart failure, liver cirrhosis, hypertension, and renal failure.
17. A method for monitoring a patient, the method comprising
- administering the mesoscale nanoparticle composition of claim 1 to the patient, wherein the mesoscale nanoparticle composition comprises an imaging agent; and
- imaging the administered mesoscale nanoparticle composition.
18. (canceled)
19. The method of claim 15, wherein the administered mesoscale nanoparticle composition demonstrates selective targeting of kidneys of the patient such that concentration of the mesoscale nanoparticle composition in the kidneys is at least 1.5 times greater than concentration of the mesoscale nanoparticle composition in any of the heart, lung, liver, or spleen of the patient from 3 days to 2 months following administration.
20-40. (canceled)
41. The mesoscale nanoparticle composition of claim 1, wherein the targeted chemotherapeutic is doxorubicin, sorafenib, or sunitinib.
42. The mesoscale nanoparticle composition of claim 1, wherein the metabolic targeting therapeutic is STF-31, CPI-613, or Fasentin.
43. The mesoscale nanoparticle composition of claim 1, wherein the TGFbeta inhibitor is LY2157299, SD-208, or SB505124.
44. The mesoscale nanoparticle composition of claim 1, wherein the Nf kappa B inhibitor is Pyrrolidine dithiocarbamate, quinazoline, BMS-345541, or BAY-11-7085.
45. The mesoscale nanoparticle composition of claim 1, wherein the therapeutic agent is encapsulated within the surface coating.
46. The mesoscale nanoparticle composition of claim 1, wherein the therapeutic agent is covalently bound to the surface coating.
47. The mesoscale nanoparticle composition of claim 1, wherein the fluorescent molecule is within the core.
48. The mesoscale nanoparticle composition of claim 1, wherein a radiolabel is attached to the nanoparticle.
Type: Application
Filed: Sep 20, 2019
Publication Date: Jul 16, 2020
Applicant: Memorial Sloan Kettering Cancer Center (New York, NY)
Inventors: Daniel A. HELLER (New York, NY), Ryan M. WILLIAMS (New York, NY)
Application Number: 16/578,250